The invention relates to probes suitable for ultrastructural localization by electron microscopy of receptors exposed at cell surfaces or present in intracellular membranes or other subcellular structures.
Knowledge of the architecture of cellular material is increasingly important to the understanding of biological systems. For example, characterization of the ultrastructural architecture of cell membranes is essential for a complete understanding of membrane function. New information concerning membrane function may provide the basis for novel drug design, therapies and diagnostic procedures.
A number of different approaches have been developed to obtain information concerning the number and localization of specific cellular components. Currently, cytochemical, immunocytochemical and radioligand techniques are the most widely employed to visualize the structure and dynamic events of membranes prior to and subsequent to some physiologic stimulus or cellular function. Each of these methods presents certain advantages and limitations but, particularly by combining the various methods, much information pertinent to the problem of membrane structure and function can be obtained.
Recently discovered techniques make EM a more useful tool for mapping cellular material. It is currently possible to purchase heavy-metal containing probes which have a large targeting protein, such as an antibody, a lectin, avidin or the like, to provide selectivity.
Colloidal gold particles are known to be useful as labels in EM studies as described by M. Horisberger in "Colloidal Gold as a Tool in Molecular Biology," TIBS (November 1983), pp. 395-397 and J. DeMey in "Colloidal Gold as a Marker and Tracer in Light and Electron Microscopy," E.N.S.A. Bulletin 14:54-66 (1984). Hicks and Molday in "Localization of Lectin Receptors on Bovine Photoreceptor Cells Using Dextran-Gold Markers," Invest. Opthalmol. Vis. Sci. 26:1002-1013 (1985), have observed that dextran derivatized with ethylenediamine readily binds to colloidal gold particles and can be used to form ligand-dextran-gold conjugates. U.S. Pat. No. 4,452,773 of Molday suggests that dextran can be used to coat colloidal sized iron particles which can be covalently bonded to antibodies for labeling cells or other biological materials.
Other patents in this field include U.S. Pat. No. 4,035,361 (Yen et al.) which discusses the production of polymeric microspheres with an attached lectin or antibody. Margel et al. discuss the formation of microspheres in U.S. Pat. No. 4,552,812. The patents of DeMey et al., U.S. Pat. Nos. 4,420,558 and 4,446,238 discuss the use of receptor-specific gold probes for bright field light microscopy. U.S. Pat. No. 4,313,734 (Leuvering) mentions the use of gold sols which are covered on the surface with antibodies as markers for EM, but primarily concerns the application of dispersed metal sol particles to determine the quantity of affinity bound components. Another assay method, that involves a labeled form of a ligand, is described in U.S. Pat. No. 4,256,725 (Rutner et al.).
It is known that Na,K-ATPase actively transports K+ and Na+ ions across plasmalemmal surfaces and therefore has a central role in the ionic and osmotic homeostasis of cells. Cytochemical, immunocytochemical and autoradiographic methods have previously been used to localize Na,K-ATPase. Ouabain is a selective inhibitor of Na,K-ATPase activity.
While existing immunogold markers can be used to label cellular material by attachment at receptor sites, methods based on cytochemical reaction products or radiography have severe limitations for EM visualization. Radioactive ligands used in autoradiography cannot be depended on to survive the washing, fixation and dehydration steps that are required to prepare biological materials for EM. For example, radiolabeled ouabain washes away under such conditions.